Intracellular Phosphoflow Cytometry of Acute Myeloid Leukemia Patient-Derived Xenotransplants

To adapt and resist approved treatments, acute myeloid leukemia (AML) cells activate specific molecular pathways that lead to changes in gene expression, protein levels and activity. In this protocol, an approach is reported to explore targets phosphorylated downstream of oncogenic signaling in AML: p-STAT5 (Tyr694), p-4EBP1 (Thr37/46), p-RPS6 (Ser240/244), and p-ERK1/2 (Thr202/Tyr204). This method enables the assessment of how these pathways-major regulators of stemness maintenance, immune evasion, protein synthesis, and adaptation to oxidative and metabolic stress-are modulated by one or more tested compounds in bone marrow cells harvested from live mice by aspiration before and after the treatment phase. This minimally invasive method preserves cell integrity and reduces stress compared to bone-crushing techniques, which can induce damage and potentially affect experimental outcomes. To optimize intracellular antibody staining for flow cytometric analysis, a protocol was developed using paraformaldehyde fixation and methanol permeabilization. This approach ensures high staining precision and minimizes background noise, enabling reliable detection of intracellular signaling markers. One of the main advantages of this protocol is the development of a multiparametric antibody panel, allowing for simultaneous assessment of the four pathways within the same sample. Using a next-generation spectral flow cytometer with high sensitivity, dynamic shifts in pathway activation were observed depending on treatment conditions compared to pretreatment baseline levels in the same mice. This methodology enables precise in vivo analysis of signaling pathway modulation in patient-derived xenograft bone marrow samples without requiring euthanasia of the animals, providing valuable insight into the adaptive mechanisms of AML cells, and can guide the evaluation of therapeutic strategies aimed at targeting these pathways to overcome resistance.